Abstract
Drug-induced hepatotoxicity is one of the major reasons cited for drug withdrawal. Therefore, it is of extreme importance to detect human hepatotoxic candidates as early as possible during the drug development process. In this study, we aimed to enhance hepatocyte functions such as CYP gene expression in HepG2 cells, one of the most extensively used cell lines in evaluating hepatotoxicity of chemicals and drugs. We found that zebularine, a potent inhibitor of DNA methylation, remarkably upregulates the expression of CYP genes in HepG2 cells. In addition, we revealed that the upregulation of CYP gene expression by zebularine was mediated through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). Furthermore, HepG2 cells treated with zebularine were more sensitive than control cells to drug toxicity. Taken together, our results show that zebularine may make HepG2 cells high-functioning and thus could be useful for evaluating the hepatotoxicity of chemicals and drugs speedily and accurately in in-vitro systems. The finding that zebularine upregulates CYP gene expression through DNMT1 and PKR modulation sheds light on the mechanisms controlling hepatocyte function and thus may aid in the development of new in-vitro systems using high-functioning hepatocytes.
Highlights
The liver is essential for maintaining normal physiology and homeostasis of the body
We found that zebularine upregulated the expressions of cytochrome P450 (CYP) genes more potently than 5-aza-dC or RG108 did, due to the differences between the actions of zebularine and those of 5-aza-dC and RG108; zebularine inhibits both DNA methyltransferase 1 (DNMT1) and PKR, whereas 5-aza-dC and RG108 inhibit DNMT1 but not PKR
By analyzing the mechanism by which zebularine upregulates CYP gene expression in HepG2 cells, we found that expression levels of CYP genes were increased by the inhibition of DNMT1 activity and by the inhibition of PKR activity
Summary
The liver is essential for maintaining normal physiology and homeostasis of the body. In HepG2 cells, epigenetic factors are known to play a role in regulating the expression of several genes involved in essential liver processes such as xenobiotic metabolism and steroid biosynthesis. Inhibitors of DNA methylation rapidly reactivate the expression of genes that have undergone epigenetic silencing[8], little is known about the impact of zebularine on gene expression in HepG2 cells or how it can be used in in-vitro assays. We found that HepG2 cells treated with zebularine are highly susceptible to xenobiotic cytotoxicity compared with controls These results will be useful for generating high-functioning HepG2 cells and may contribute to the development of a simple and rapid method of assaying a substance’s potential for liver damage
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